In the field of biotechnological pharmaceuticals developed by applying genetic recombination technologies, particularly the market for antibody pharmaceuticals has grown rapidly in recent years while there have been raised concerns about their loading on medical expenses; so there has always been a demand for developing a technique for producing biotechnological pharmaceuticals that enable more efficient protein production and are more low-cost than conventional ones.
Examples of hosts that have been used for protein production using genetic recombination technologies include animal cells, yeast, and Escherichia coli. E. coli and the like are capable of producing a protein as a product of interest with low costs, but are unfit for glycoprotein production because no post-translational modification such as sugar-chain modification can be achieved in such microorganisms. In addition, E. coli has a tendency to form an inclusion body containing a produced protein, and thus has a disadvantage in that, in order to obtain a protein as a product of interest, a solubilization process is further required after synthesis, thereby causing heavy workload.
Particularly in the case of glycoproteins such as antibodies, an added sugar chain has an influence on the water solubility of a protein as a product of interest, its resistance to a protease, its tissue-targeting capability, and its biological activity; thus, there has been a need for production technologies using animal cells from higher eukaryotes, and these technologies have advanced considerably in recent years. Under these circumstances, many current antibody pharmaceuticals are produced using Chinese hamster ovary (CHO) cells, and optimizing production processes for such pharmaceuticals is still an important challenge.
Proteins secreted extracellularly from eukaryotic cells including mammalian cells are synthesized in the endoplasmic reticulum which is intracellular organelle divided by endomembranes. The endoplasmic reticulum is broadly classified into the following two types: a rough endoplasmic reticulum studded on its surface with ribosomes which are machines for protein synthesis composed of a RNA-protein macrocomplex, and a smooth endoplasmic reticulum with no ribosomes, but the detailed mechanism of formation of the rough endoplasmic reticulum has been yet to be elucidated.
In the living body, there are professional secretory cells specialized in secreting particular proteins, and these professional secretory cells have highly developed rough endoplasmic reticulum which are considered to enable highly efficient protein production. Examples of such professional secretory cells include fibroblasts secreting collagen, and pancreatic exocrine secretory cells secreting a group of digestive enzymes. As compared to those professional secretory cells, rough endoplasmic reticulum such as CHO cells and HEK293 cells, which are now often used for genetically engineered protein production, are problematic in that they are present only in a very small amount and are inferior in secretory activity.
In the process of production of biotechnological pharmaceuticals using genetic recombination technologies, the genes of a protein as a product of interest are under the control of a promoter showing high transcription activity in an expression vector, and are presumed to express their mRNA at a high level. However, even under these conditions, the mRNA level is often not correlated with the expressed protein amount per se, and one of the factors for this may be due to low efficiency of mRNA translation on the endoplasmic reticulum membranes.
These observation suggest that there may be room for further enhancement of the protein synthetic capacity in the aforementioned cells that are now widely used for genetically engineered protein production, if mRNA can be provided in a more appropriate manner to be used to the machines for translation on the endoplasmic reticulum membranes like in the case of fibroblasts.
It is known that fibroblasts permanently secreting collagen constantly express a high level of collagen protein-encoding mRNAs, the majority of which is detected on the endoplasmic reticulum, a place of biosynthesis of the secretory proteins (Non-patent Literature 1). However, its more localization of the collagen mRNA on the endoplasmic reticulum is not sufficient to activate collagen synthesis, but the formation of a polysome having high translation efficiency is also needed for activated synthesis.
The previous analyses made by the present inventors revealed that the mRNAs for some types of proteins, including collagen genes, have a tendency to form a polysome in which multiple ribosomes, machines for protein synthesis are associated to each other (Patent Literature 1, Non-patent Literature 2). This finding led to the conjecture that the reason why, in the process of production of biotechnological pharmaceuticals using genetic recombination technologies, gene transcripts encoding a protein of interest are expressed at a high level and nevertheless the protein is synthesized or secreted only in a small amount, it may be because in used cells, mRNA is not provided to the machines for translation on the endoplasmic reticulum membranes in an easy-to-use form.